In the germination of seeds of cereal plants, starch degradation is an important metabolic process. Starch is the primary source of carbon and energy for cereal seedlings until they become autotrophic. Degradation of cereal starches in cereal seedlings is a result of the concerted action of several enzymes including α-amylase, β-amylase, debranching enzyme and α-glucosidase. It has been observed that during the early stages of starch hydrolysis in germinating cereal seeds, α-amylase is the most important enzyme and α-glucosidase is the second most important enzyme to the seedlings starch degradation processes.
Starch degradation processes are important for other reasons besides the viability and vigor of cereal seedlings. Many food processes involve the conversion of starch from cereal plants for food or other uses. It is known that α-glucosidase accelerates the initial hydrolysis of starch granules in the presence of α-amylase. In vitro, barley α-glucosidase can hydrolyze native starch granules at a rate comparable to α-amylase. In addition, the two enzymes act synergistically in the starch degradation process.
For food production applications, and in other industrial processes to produce or process starches from cereals, thermal stability of enzymes becomes an important criteria. For example, the thermal stability of α-glucosidase is important because the conversion of barley starch to fermentable sugars during the industrial production of ethanol, as in brewing or in fuel ethanol production, typically takes place at temperatures of 65 to 73° C. The thermal lability of many native barley α-glucosidase enzymes results in either reduced efficiency of starch break down at the higher temperatures used for starch gelatinization, or requires that the starch be cooled to a more favorable temperature for enzymatic hydrolysis after the starch is gelatinized.
Significant research has occurred on barley α-glucosidase in the last few years. In fact, the native barley gene for α-glucosidase has been sequenced, cloned, and the amino acid sequence of the resulting expressed enzyme has been determined. The DNA sequence of the native cDNA and the amino acid sequence of the protein are fully described in U.S. Pat. No. 5,763,252, the disclosure of which is incorporated herein by reference.
While the full sequence of barley α-glucosidase is known, many critical details about the structure and function of the enzyme are still uncharacterized. No crystal structure has been determined for any α-glucosidase of the glucosyl hydrolase family, making it much more difficult to intelligently select targets for mutagenesis. It is known that the α-glucosidase genes from various plants do have variations in their thermostability, but the rationales and reasons behind those differences are obscure. The lack of thermostable α-glucosidases has been a limitation in the industrial use of α-glucosidase enzymes to replace or supplement α-amylases in industrial hydrolysis systems. Thus the need exists for more thermostable α-glucosidases which can be used for a wide variety of industrial and food preparation purposes such as specifically brewing and fuel ethanol production.